Fuchs and B

Fuchs and B. species is responsible for the main malaria burden in sub\Saharan Africa giving rise to the most lethal cerebral form of the disease.[ 1 , 2 ] Infections with predominate outside of Africa[ 3 , 4 ] and can persist by the formation of hypnozoites that remain in the host’s liver to relapse after months or even years of dormancy.[ 5 , 6 , 7 ] The remaining three human\pathogenic species cause morbidity of geographically more localized significance. [ 8 , 9 ] Growing case numbers and increasing resistance of parasite strains keep an unrelieved pressure on research efforts to discover novel drugs and unexploited targets.[ 10 , 11 ] Recently, we identified the sole l\lactate/H+ transporter from spp., FNT\type transporters are present in other protozoal parasites such as parasites at nanomolar concentrations. [13] Treatment with sublethal doses of MMV007839 forced a resistance mutation in the PfFNT\encoding gene resulting in an exchange of Gly107 to serine on the protein level, PfFNT G107?S. [13] The mutation decreases the affinity of MMV007839 to PfFNT Lauric Acid G107?S likely due to a clash Lauric Acid of the small molecule’s phenyl\hydroxyl moiety with the introduced serine sidechain in the target protein. A serine hydroxy group, however, bears the potential for exploitation by using the hydrogen\bond as an interaction site with an inhibitor. Indeed, creating the new class of pyridine\substituted pentafluoro\3\hydroxy\pent\2\en\1\ones lacking a hydroxyl at the aromatic ring yet carrying a single nitrogen atom as a hydrogen bond acceptor site (BH297, BH267.meta; Figure?1?A), re\established nanomolar PfFNT inhibition irrespective of the presence of the G107?S mutation. [24] Open in a separate Lauric Acid window Figure 1 FNT inhibitors and structure comparison of FNTs from human\pathogenic plasmodia. A) Structures of three inhibitors for plasmodial FNT\type l\lactate transporters: MMV007839, BH297, and BH267.meta. B) Protein sequence similarity and identity of the human\pathogenic plasmodial FNTs (Pf: species (PfFNT, PvFNT, PmFNT, PoFNT, PkFNT), and their potent inhibition by the screening hit MMV007839 as well as its pyridine analogues BH297 and BH267.meta. We further developed a yeast\based phenotypic screening assay that allows for high\throughput assessment of FNT inhibitor efficacy. Finally, we demonstrate the suitability of the compounds for use in animal models by nanomolar inhibition of PbFNT from species that infects mice. BH267.meta exhibited particularly low mammalian cell toxicity which is a prerequisite for use. Results Plasmodial FNTs exhibit conserved transport paths and putative inhibitor binding sites We started out by using a bioinformatics approach for protein sequence comparisons of PfFNT, PvFNT, PmFNT, PoFNT, PkFNT (full alignment shown in Figure?S1 in the Supporting Information). The sequence length of the plasmodial FNT proteins varied by only by one amino acid comprising 308 or 309?positions. Total pair\wise sequence identity ranged from 72.4?%, when comparing PfFNT with PmFNT, to 90.5?% for PvFNT and PkFNT (Figure?1B). Considering additional sequence positions that hold physicochemically related amino acid residues led to similarity scores between 84.1?% (PfFNT / PmFNT) and 95.4?% (PvFNT / PkFNT; Figure?1B). The amino acid positions that make up the most relevant sites in the sequence were even fully conserved, namely the /K substrate selectivity filter and the two FNT\typical constriction sites in the transport path (Figure?S1). Next, we generated 3D structure models of TNFRSF16 the plasmodial FNT proteins based on the crystal structure of a bacterial FNT homolog (PDB ID: 3TE0). [25] Figure?1?C depicts the PfFNT model as viewed from the intracellular space into the transport path of one protomer. Lauric Acid This lower half of the transport path represents the putative FNT inhibitor binding site (orange circle) and contains the G107?S resistance mutation site (dark cyan). We projected the degree of sequence variation obtained from the sequence alignment (whiteCred scale) onto the model in order to localize the positions of deviating residues in a structural context. This revealed that amino acid differences among the spp. FNTs are restricted to the periphery (red shading), which anchors the protein in the lipid membrane, whereas the substrate transport path including the inhibitor binding site appears virtually invariant (in white). spp. FNTs.Cells were precultured overnight before treatment with the indicated compounds for 24?h. malaria burden in sub\Saharan Africa giving rise to the most lethal cerebral form of the disease.[ 1 , 2 ] Infections with predominate outside of Africa[ 3 , 4 ] and can persist by the formation of hypnozoites that remain in the host’s liver to relapse after months or even years of dormancy.[ 5 , 6 , 7 ] The remaining three human\pathogenic species cause morbidity of geographically more localized significance.[ 8 , 9 ] Growing case numbers and increasing resistance of parasite strains keep an unrelieved pressure on research efforts to discover novel drugs and unexploited targets.[ 10 , 11 ] Recently, we identified the sole l\lactate/H+ transporter from spp., FNT\type transporters are present in other protozoal parasites such as parasites at nanomolar concentrations. [13] Treatment with sublethal doses of MMV007839 forced a resistance mutation in the PfFNT\encoding gene resulting in an exchange of Gly107 to serine on the protein level, PfFNT G107?S. [13] The mutation decreases the affinity of MMV007839 to PfFNT G107?S likely due to a clash of the small molecule’s phenyl\hydroxyl moiety with the introduced serine sidechain in the target protein. A serine hydroxy group, however, bears the potential for exploitation by using the hydrogen\bond as an interaction site with an inhibitor. Indeed, creating the new class of pyridine\substituted pentafluoro\3\hydroxy\pent\2\en\1\ones lacking a hydroxyl at the aromatic ring yet carrying a single nitrogen atom as a hydrogen bond acceptor site (BH297, BH267.meta; Figure?1?A), re\established nanomolar PfFNT inhibition irrespective of the presence of the G107?S mutation. [24] Open in a separate window Figure 1 FNT inhibitors and structure comparison of FNTs from human\pathogenic plasmodia. A) Structures of three inhibitors for plasmodial FNT\type l\lactate transporters: MMV007839, BH297, and BH267.meta. B) Protein sequence similarity and identity of the human\pathogenic plasmodial FNTs (Pf: species (PfFNT, PvFNT, PmFNT, PoFNT, PkFNT), and their potent inhibition by the screening hit MMV007839 as well as its pyridine analogues BH297 and BH267.meta. We further developed a yeast\based phenotypic screening assay that allows for high\throughput assessment of FNT inhibitor efficacy. Finally, we demonstrate the suitability of the compounds for use in animal models by nanomolar Lauric Acid inhibition of PbFNT from species that infects mice. BH267.meta exhibited particularly low mammalian cell toxicity which is a prerequisite for use. Results Plasmodial FNTs exhibit conserved transport paths and putative inhibitor binding sites We started out by using a bioinformatics approach for protein sequence comparisons of PfFNT, PvFNT, PmFNT, PoFNT, PkFNT (full alignment shown in Figure?S1 in the Supporting Information). The sequence length of the plasmodial FNT proteins varied by only by one amino acid comprising 308 or 309?positions. Total pair\wise sequence identity ranged from 72.4?%, when comparing PfFNT with PmFNT, to 90.5?% for PvFNT and PkFNT (Figure?1B). Considering additional sequence positions that hold physicochemically related amino acid residues led to similarity scores between 84.1?% (PfFNT / PmFNT) and 95.4?% (PvFNT / PkFNT; Figure?1B). The amino acid positions that make up the most relevant sites in the sequence were even fully conserved, namely the /K substrate selectivity filter and the two FNT\typical constriction sites in the transport path (Figure?S1). Next, we generated 3D structure models of the plasmodial FNT proteins based on the.